The long-term objective of the proposed work is the elucidation of the mechanism of DNA replication in animal mitochondria, and its relationship to mitochondrial mutagenesis and human disease. A combined approach of current methods in biochemistry and molecular genetics is being pursued to study the mechanism, structure and physiology of the major replicative enzyme in mitochondria, DNA polymerase gamma (pol gamma), and the specific roles of the essential replication accessory protein, mitochondrial single-stranded DNA-binding protein. Structure-function studies of pol gamma will involve comparative analyses of native and altered forms, to elucidate the role of the small subunit in enzyme function and to dissect functional domains in both the catalytic and accessory subunits. Physiological studies will focus on expanding the development of a transgenic fly model to examine the relationship between pol gamma function, mitochondrial DNA replication fidelity, aging and disease. The control of animal cell reproduction during normal development, and the loss of control during cancerous development, is of central importance in the processes of human growth, aging, and disease. Mitochondrial biogenesis proceeds in parallel with cell proliferation, but it is neither tightly coupled to mitochondrial DNA replication nor to the cell cycle. Nevertheless, because both the DNA content of the mitochondrion and the number of mitochondria in cells remain relatively constant, specific regulatory mechanisms are likely required to couple mitochondrial DNA replication and biogenesis to nuclear DNA replication and cell division. A detailed analysis of the key enzyme involved in mitochondrial DNA replication, DNA polymerase gamma, will represent a major contribution toward an eventual understanding of mitochondrial biogenesis and function in normal and diseased tissues. A variety of mitochondrial DNA diseases have recently been documented. This, and an increased recognition that antiviral and antitumor drugs frequently affect mitochondrial DNA function, and in particular, the activity of po1 gamma, demonstrate a critical need for an in-depth understanding of this essential cellular DNA polymerase.